CN107446954A - A kind of preparation method of SD rat T cells deleting genetic model - Google Patents
A kind of preparation method of SD rat T cells deleting genetic model Download PDFInfo
- Publication number
- CN107446954A CN107446954A CN201710646278.XA CN201710646278A CN107446954A CN 107446954 A CN107446954 A CN 107446954A CN 201710646278 A CN201710646278 A CN 201710646278A CN 107446954 A CN107446954 A CN 107446954A
- Authority
- CN
- China
- Prior art keywords
- rat
- lck
- cells
- rats
- ivt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 45
- 230000002068 genetic effect Effects 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 241000700159 Rattus Species 0.000 claims abstract description 76
- 101150028321 Lck gene Proteins 0.000 claims abstract description 27
- 108091033409 CRISPR Proteins 0.000 claims abstract description 19
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 12
- 239000012634 fragment Substances 0.000 claims abstract description 10
- 108020004414 DNA Proteins 0.000 claims description 34
- 108020004999 messenger RNA Proteins 0.000 claims description 22
- 238000003209 gene knockout Methods 0.000 claims description 17
- 210000004027 cell Anatomy 0.000 claims description 15
- 238000000338 in vitro Methods 0.000 claims description 10
- 238000012408 PCR amplification Methods 0.000 claims description 8
- 208000033040 Somatoform disorder pregnancy Diseases 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 241000699670 Mus sp. Species 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 4
- 239000000969 carrier Substances 0.000 claims description 3
- 230000002950 deficient Effects 0.000 claims description 3
- 108700029318 rat female Proteins 0.000 claims description 3
- 230000004083 survival effect Effects 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 210000000287 oocyte Anatomy 0.000 claims description 2
- 210000005259 peripheral blood Anatomy 0.000 claims description 2
- 239000011886 peripheral blood Substances 0.000 claims description 2
- 238000013467 fragmentation Methods 0.000 claims 1
- 238000006062 fragmentation reaction Methods 0.000 claims 1
- 101100074246 Rattus norvegicus Lck gene Proteins 0.000 abstract description 6
- 238000011161 development Methods 0.000 abstract description 5
- 201000010099 disease Diseases 0.000 abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
- 238000000034 method Methods 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 4
- 238000010171 animal model Methods 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 238000012217 deletion Methods 0.000 abstract 1
- 230000037430 deletion Effects 0.000 abstract 1
- 238000012239 gene modification Methods 0.000 abstract 1
- 230000005017 genetic modification Effects 0.000 abstract 1
- 235000013617 genetically modified food Nutrition 0.000 abstract 1
- 101150044508 key gene Proteins 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- 238000013518 transcription Methods 0.000 description 10
- 230000035897 transcription Effects 0.000 description 10
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 7
- 229910001868 water Inorganic materials 0.000 description 7
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 5
- 230000001186 cumulative effect Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000013589 supplement Substances 0.000 description 4
- 238000010354 CRISPR gene editing Methods 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 230000007969 cellular immunity Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 210000001541 thymus gland Anatomy 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 2
- 240000003186 Stachytarpheta cayennensis Species 0.000 description 2
- 235000009233 Stachytarpheta cayennensis Nutrition 0.000 description 2
- 108091028113 Trans-activating crRNA Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011552 rat model Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 102000005869 Activating Transcription Factors Human genes 0.000 description 1
- 108010005254 Activating Transcription Factors Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000180579 Arca Species 0.000 description 1
- 108091079001 CRISPR RNA Proteins 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101001047681 Homo sapiens Tyrosine-protein kinase Lck Proteins 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 206010042573 Superovulation Diseases 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 102100024036 Tyrosine-protein kinase Lck Human genes 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000009405 line breeding Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012372 quality testing Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of preparation method of SD rat T cells deleting genetic model, belong to genetic engineering and genetic modification technique field.The present invention realizes the specific knockdown of the key gene Lck to control rat T cells development using CRISPR Cas9 systems in rats, obtains SD rat T cells deleting genetic models.The present invention determines 2 specific target practice sites for being directed to rat Lck genes first, and being experimentally confirmed it has very high shear efficiency；The present invention is practiced shooting using 2 sgRNA for rat Lck genes simultaneously, and obtains the knockout rat of large fragment deletion, and the gene that on the one hand can ensure to be practiced shooting can thoroughly lose function, and still further aspect can also facilitate subsequent detection.The SD rat animal models for building obtained T cell missing have very great meaning in immune and disease research.
Description
Technical field
The present invention relates to a kind of preparation method of SD rat T cells deleting genetic model, belong to genetic engineering and heredity is repaiied
Adorn technical field.
Background technology
Lck (Lymphocyte-specific protein tyrosine kinase) activation is to start TCR signals to lead to
The key factor on road, it all plays critical effect in the development of thymus gland and the differentiation of T cell and development, so big
Lck genes are knocked out in mouse, you can to obtain the rat animal model of T cell missing.At present, the method for conventional gene knockout has
Three kinds：1. Zinc finger nuclease (ZFNs)；2. activating transcription factor sample effector nuclease (TALENs)；3.CRISPR-Cas9.Zinc
Possibility prospects of the finger nuclease ZFNs in Plant Genome fixed point transformation, but because ZFNs synthesis assembling technical difficulty is big,
Common laboratory is difficult to carry out, and ZFNs is easy to carry out Non-specific cleavage to genome, or to target spot DNA cutting efficiencies
It is low, it is limited always enters practical application.Compared to traditional Zinc finger nuclease technology, TALENs has unique advantage：If
Meter is simpler, and specificity is higher.But there is certain cytotoxicity, module assembled process is cumbersome, generally require ask assist in it is outer
Bao Zheng department.
CRISPR refers to cluster, rule short palindrome repetitive sequence (Clustered Regularly
Interspersed Short Palindromic Repeats).CRISPR-Cas9 systems are mainly made up of three parts,
It is Cas9 albumen, precursor CRISPR RNA (pre-crRNA) and trans-activating crRNA respectively
(tracrRNA), CRISPR-Cas9 identifies specific DNA sequence dna, carries out specific site cutting and causes double-strand DNA cleavage, is not having
Under conditions of having masterplate, the connection of non-homogeneous restructuring end occurs, causes frameshift mutation, causes gene knockout.CRISPR-Cas9 systems
Small crRNA guiding is leaned in the identification to specific position of uniting, and CRISPR areas can be made up of a series of crRNA, Mei Gezhen
There was only tens bases to the crRNA of specific position, whole carrier is smaller, relative to ZFNs and TALENs carriers, more holds
Easily structure.
T cell derives from the multipotential stem cell of marrow.In embryonic period, embryonic phase and nascent phase, a part of multipotential stem cell in marrow
Or pre-T cell is moved in thymus gland, the differentiation and maturation under the induction of thymin, turn into immunocompetent T cell.It is ripe
Thymus dependent area of the T cell through blood distribution to peripheral immune organ settle down, and can be through lymphatic vessel, peripheral blood and tissue fluid etc.
Recycled, play the function such as cellular immunity and immunological regulation.The recycling of T cell is advantageous to contact extensively and entered in vivo
Antigenic substance, booster immunization response, longer-term keep immunological memory.There are many different marks on the cell membrane of T cell, mainly
It is surface antigen and surface receptor.These surface markers are all incorporated in the huge protein molecular on cell membrane.T cell is that lymph is thin
The key component of born of the same parents, it has various biological function, and such as direct killing target cell, auxiliary or the B cell that suppresses produce antibody, right
The former responsing reaction of specific antigen and mitogenesis and produce cell factor etc., be in body for resist disease infection,
Tumour and the brave fighter formed.Immune response caused by T cell is cellular immunity, and the effect form of cellular immunity mainly has two
Kind：Combined with target cell specificity, destroy target cell membrane, direct killing target cell；Another kind is release lymphokine, is finally made
Immunological effect expands and enhancing.
T cell plays an important role in some immune responses to catch, determines the effect of T cell, understands it
Function helps to illustrate duplication, the interaction of mechanism of causing a disease of internal T cell and cause of disease.Build the animal mould of T cell missing
Type is immune with having very great meaning in disease research.
The content of the invention
A kind of the present invention is intended to provide preparation method of SD rat T cells deleting genetic model.
To achieve these goals, the technical solution adopted in the present invention is：
A kind of preparation method of SD rat T cells deleting genetic model, comprises the following steps：
Comprise the following steps：
1) target sequence is determined：Two target sequences are respectively：sgRNA1：5 '-GCCCAAGTCTCCATCATGGG AGG-3 ',
sgRNA2：5‘-GACCCACTGGTCACCTATGA GGG-3’；
2) primer is designed：The sgRNA target sequences obtained according to step 1) design 3 primers, are respectively：Rat-LCK-IVT-
1：5‘-TTAATACGAC TCACTATAGG GGCCCAAGTC TCCATCATGG GGTTTTAGAG CTAGAAATAG CAAG-
3 ', Rat-LCK-IVT-2：5‘-TTAATACGAC TCACTATAGG GGACCCACTG GTCACCTATG AGTTTTAGAG
CTAGAAATAG CAAG-3 ', Rat-LCK-IVT-3：5‘-AAAAAAGCAC CGACTCGGTG CC-3’；
3) double chain DNA fragment is obtained：Rat-LCK-IVT-1 in step 2) and Rat-LCK-IVT-3 is matched, Rat-LCK-
IVT-1 and Rat-LCK-IVT-3 pairings, using pX330 plasmids as template, enter performing PCR amplification；Pcr amplification product is purified, obtained
Double chain DNA fragment；
4) sgRNA mRNA are obtained：It is pure after transcribing in vitro using the double chain DNA fragment that step 3) obtains as template
Change, obtain sgRNA1 mRNA and sgRNA2 mRNA；
5) Cas9 mRNA are obtained and are purified：Cas9 carriers are linearized through AgeI, purified after transcribing in vitro, i.e.,
Obtain Cas9 mRNA；
6) SD rat T cells deleting genetic models are obtained：The Cas9 mRNA that step 5) obtains are obtained with step 4)
After sgRNA1 mRNA and sgRNA2 mRNA mixing, it is injected into rat fertilized egg cell, by the fertilized oocyte of survival
Enter false pregnancy SD rat female mice fallopian tubals；After false pregnancy SD rats are given a birth, Founder rats are obtained；
Founder rats and wild type SD rats are returned, select F1Continue to be returned with wild type SD rats for heterozygote,
Repetition picks out heterozygote from offspring and is used to be returned, and heterozygote selfing is selected in backcross progeny, and self progeny obtains after testing
The homozygote arrived carries out Immunophenotype analysis, and complete T cell deficient is SD rat T cells deleting genetic models.
In step 1), target gene Lck DNA sequence dna is found in genome database, then using online software
CRISPOR respectively selects target sequence of 1 specific position as sgRNA in the target site exon3 and exon4 of rat Lck genes
Row, have higher gene knockout efficiency.
In step 6), Cas9 mRNA, sgRNA1 mRNA, sgRNA2 mRNA injection concentration are 50-100ng/ul.
Immunophenotype analysis concrete operations are carried out in step 6) is：Extract the homozygous periphery of LCK gene knockout SD rats
Blood, by Flow cytometry, whether its T cell has lacked completely.
T cell is marked using T cell specific antibody, then by Flow cytometry its T cell whether
Lack completely.
The present invention realizes the key to control rat T cells development using CRISPR-Cas9 systems in rats first
Gene Lck specific knockdown, obtain SD rat T cells deficient animal models.Beneficial effects of the present invention are as follows：：1.SD is big
Mouse line breeding, zoopery are widely used in biomedicine field, but SD rat genes knock out model and still extremely lacked,
More Lck gene knockouts are realized in SD rats first and obtain T Lymphocyte depletions without immune deficiency genetic model, the present invention
Stable genetic model；2. 2 specific target practice sites for being directed to rat Lck genes are determined first, its tool is experimentally confirmed
There is very high shear efficiency；3. practiced shooting first using 2 sgRNA for rat Lck genes simultaneously, and obtain large fragment
The knockout rat of missing, the gene that on the one hand can ensure to be practiced shooting can thoroughly lose function, and still further aspect can also
Facilitate subsequent detection；4. knocking out Lck genes using CRISPR-Cas9 systems in rat at home and abroad to still belong to the first time, have very high
Original and very important basic research and actual application value, also for gene knockout in rats work development provide
Good demonstration.
Brief description of the drawings
Fig. 1 is sgRNA transcription product quality testing figures；
Fig. 2 is that Founder P of Rats CR detects electrophoretic band figure；
Fig. 3 is Lck gene knockout target practices site schematic diagram and Lck gene knockout Founder rat sequencing sequence figures；
Fig. 4 is Lck gene knockout SD rat peripheral haemocyte flow cytometer testing result figures.
Embodiment
With reference to specific embodiment, the present invention is described in further detail.
Embodiment 1
First, according to CRISPR/Cas9 system principles, CRISPOR Photographing On-line softwares (http is used://
Crispor.tefor.net/crispor.cgi), in target site exon3 (the exon ID of rat Lck genes：
) and exon4 (exon ID ENSRNOE00000091030：ENSRNOE00000091075 1 specific position is respectively selected in) to make
For sgRNA target sequence, the two target sequences are respectively：sgRNA1(SEQ ID NO.1)：sgRNA1：5‘-
GCCCAAGTCTCCATCATGGGAGG-3’；sgRNA2(SEQ ID NO.2)：5-GACCCACTGGTCACCTATGAGGG-3.
2nd, 3 primers are ordered in the Li Ge Bioisystech Co., Ltd of Shanghai hundred：Rat-LCK-IVT-1(SEQ ID
NO.3)：5‘-TTAATACGAC TCACTATAGG GGCCCAAGTC TCCATCATGG GGTTTTAGAG CTAGAAATAG
CAAG-3 ', Rat-LCK-IVT-2 (SEQ ID NO.4)：5‘-TTAATACGAC TCACTATAGG GGACCCACTG
GTCACCTATG AGTTTTAGAG CTAGAAATAG CAAG-3 ', Rat-LCK-IVT-3 (SEQ ID NO.5)：5‘-
AAAAAAGCAC CGACTCGGTG CC-3’。
3rd, expanded by PCR, obtain the double-stranded DNA with T7 promoters and (T7 promoter sequences are added into drawing for synthesis
Thing sequence 5 ' end, by PCR amplification can obtain the double-stranded DNA with T7 promoters)：Wherein, PCR reaction systems are：
Template is pX330 DNAs (being purchased from Addgene), adds 10ng；Sense primer Rat-LCK-IVT-1 (10 μM) and Rat-
LCK-IVT-2 (10 μM) is separately added into 2 μ l；Downstream universal primer Rat-LCK-IVT-3 (10 μM) is separately added into 2 μ l；2×Taq
Master Mix (being purchased from vazyme, P111-01) add 25 μ l；Supplement H2O is to μ l (the double-stranded DNA sequences that PCR is obtained of cumulative volume 50
Row are as shown in SEQ ID NO.8 and SEQ ID NO.9).PCR response procedures are：94 DEG C, 5min；94 DEG C, 30s, 60 DEG C, 30s,
72 DEG C, 30s；72 DEG C, 10min；Period is 30 times.After PCR EPs (end of program), invitrogen PCR QIAquick Gel Extraction Kits are used
(invitrogen, K220001), purified pcr product.
4th, in-vitro transcription obtains sgRNA mRNA.In-vitro transcription kit uses NEB kits (NEB, E2050S), in detail
Thin reactions steps are as follows：Required according to kit, prepare reaction system, sequentially add the μ l of NTP Buffer Mix 10；Template
DNA 1μg；T7RNA Polymerase Mix 2μl；Supplement H2O to the μ l of cumulative volume 30；It is placed in 37 DEG C of reactions to be incubated 4 hours, obtains
External transcription product is obtained, obtained sgRNA transcription products run cementing fruit as shown in figure 1,1：marker；2：Purpose band is
sgRNA1；3：Purpose band is sgRNA2；To specifications using Ambion RNA purification kits (Ambion, AM1909)
Recovery band is asked to obtain sgRNA1 mRNA and sgRNA2 mRNA.
5th, Cas9 in-vitro transcription template DNAs are obtained.Use high-purity plasmid extraction kit (Tiangeng biochemical technology (north
Capital) Co., Ltd, DP116) extraction Cas9 vector plasmid DNAs, then use AgeI restriction enzymes using following system
(NEB, R3552S) digested plasmid DNA：DNA：10μg；CutSmart Buffer：20μl；AgeI restriction enzymes：2μ
l；Supplement H2O to the μ l of cumulative volume 200.The mouth of pipe is sealed using sealing compound, is subsequently placed in 37 DEG C of incubator reaction overnights.Pass through agar
Sugared detected through gel electrophoresis digestion products, single band is presented after digestion products electrophoresis, shows that vector plasmid DNA linearisation is completed.
Digestion products are purified using DNA QIAquick Gel Extraction Kits (TIANGEN Biotech (Beijing) Co., Ltd., DP205), that is, obtain Cas9 bodies
Outer transcription templates DNA.
6th, in-vitro transcription obtains Cas9 mRNA.In-vitro transcription kit uses invitrogen kits
(invitrogen, AMB1345-5), detailed reaction step are as follows：Required according to kit, prepare reaction system, sequentially add
T7 2*NTP/ARCA 10μl；10*T7 Reaction Buffer 2μl；The μ g of template DNA 1；T7 Enzyme Mix 2μl；Mend
H2O (invitrogen, 10977015) is filled to the μ l of cumulative volume 20；It is placed in 37 DEG C of reactions to be incubated 2 hours, then adds 1 μ l
TURBO DNase, after fully mixing, it is placed in 37 DEG C of reactions and is incubated 15min；Next again into above reaction product according to
Lower order prepares tailings reactions system, the μ l of mMESSAGE mMACHINE T7 Ultra reaction products 20；H2O
(invitrogen, 10977015) 36 μ l；5*E-PAP Buffer 20μl；25mM MnCl2 10μl；ATP Solution 10
μl；The μ l of E-PAP 4, after fully mixing, it is placed in 37 DEG C of reactions and is incubated 45min.After obtaining in-vitro transcription product, use
Ambion RNA purification kits (Ambion, AM1909) recovery Cas9 mRNA.
7th, sgRNA mRNA and the Cas9 mRNA obtained injects SD rat fertilized egg cells by micro-injection system.It is logical
Cross and hormone is injected to 4 week old SD rats female mices, fertilized egg cell is obtained after super ovulation and mating.By sgRNA1 mRNA,
It is equal that sgRNA2 mRNA and Cas9 mRNA using the ultra-pure water (invitrogen, 10977015) without RNase are diluted to final concentration
For 50ng/ μ l, be injected into after mixing by micro-injection system in SD rat fertilized egg cells, be placed in incubator (37 DEG C, CO2
Concentration is after 5%) cultivating 1 hour, to select the cell of survival, be implanted into false pregnancy SD rat female mice fallopian tubals.Note altogether
77 fertilized egg cells are penetrated, are survived 76,3 false pregnancy female mices is transplanted, finally gives 2 Founder rats.
8th, Founder rat genes type is identified.Obtain 2 Founder rats are cut into rat-tail, extract DNA, Ran Houli
By the use of it as template DNA, enter performing PCR amplification, be using primer sequence：
LCK-F(SEQ ID NO.6)：5 '-CCAGGATTCGGAGCTGTTCA-3 ',
LCK-R(SEQ ID NO.7)：5‘-ATTTCCTACCCCACAGCTGC-3’；
Wild type DNA profiling expanding fragment length is 358bp (extension increasing sequence is as shown in SEQ ID NO.10), and PCR reacts
System is：Template is rat-tail DNA, adds 10ng；Sense primer adds 2 μ l；Anti-sense primer adds 2 μ l；2×Taq Master
Mix (being purchased from vazyme, P111-01) adds 25 μ l；Supplement H2O to the μ l of cumulative volume 50.PCR response procedures are：94 DEG C, 5min；
94 DEG C, 30s, 60 DEG C, 30s, 72 DEG C, 30s；72 DEG C, 10min；Period is 30 times.After obtaining PCR primer, 10 μ l products are taken
For running glue, compareed with wild-type amplification product, find the amplification of 2 Founder rats (being respectively designated as #1 and #2)
Fragment is compared with wild type, and missing is more, as a result as shown in Fig. 21：marker；2：Negative control H2O；3：Wild type SD is big
Mouse；4：Wild type SD rats；5：Lck knockout rats #1；6：Lck knockout rats #2.Take 2 μ l#1 and #2 rats
Pcr amplification product is used for TA clone's coupled reactions (TIANGEN Biotech (Beijing) Co., Ltd., DP205), then produces connection
Thing converts DH5 α competent cells, is incubated overnight in 37 DEG C of incubators, after it grows monoclonal, picking monoclonal is trained
Support, and sample presentation carries out sequencing identification.2 Founder rats carry out sequencing identification, and sequencing result is as shown in figure 3, result table
Bright, two chromosomes of #1 rats lack 280 and 249 bases respectively, and #2 rats item chromosome lacks 70 bases.
9th, LCK gene knockout SD rat homozygotes are obtained by being returned and being selfed.By 2 Founder rats of acquisition point
Do not mate to obtain F1 generation with wild type SD rats, screen to obtain F1 generation LCK gene knockouts with DNA sequencing technology by PCR amplifications
Rat heterozygote individual (totally 4 kinds of genotype), F1 generation heterozygote rat is continued to be returned with wild type SD rats respectively, and every time
Heterozygote knockout rat is picked out from offspring to be used to be returned, and after being returned for 6 generations, selects F6 heterozygote knockout rats
Selfing obtains F7 for rat, and further detection, can screen acquisition homozygotic individual, the Lck knockout rat moulds as built
Type.
Tenth, flow cytometry Lck gene knockout SD rat immunity phenotypes are passed through.Obtain LCK gene knockout SD rats
After homozygote, from its tail vein blood, detected by flow cytometer (BD, Canto II plus), confirmed its T cell
Missing completely, shows that Lck gene knockout SD rat models successfully construct.Detection is respectively with antibody：CD3 (Biolegend,
B223192), CD4 (Biolegend, B227587), B220 (eBioscience, 25-0460-82), CD45
(eBioscience, 48-0461-82).Detailed operating procedures are as follows：It is quiet first by the anticoagulant tube collection rat with EDTA
The μ l of arteries and veins blood 50, then using FACS buffer solution prepare suitable concn antibody mixed liquor (in final system, antibody CD3 dilution
200 times, antibody CD4 dilutes 400 times, and antibody B220 dilutes 200 times, and antibody CD45 dilutes 200 times), take 10 μ l antibody mixed liquors
Fully it is vortexed mixing with 40 μ l anti-freezings rat bloods, is subsequently placed in 4 DEG C of refrigerators and is incubated 30min.Add into the mixed liquor being incubated
Enter 500 μ l erythrocyte cracked liquids (eBioscience), being fully vortexed mixes, and then at 5min is stored at room temperature, red blood cell is fully split
Solution；1ml FACS buffer solutions are added in most backward mixed liquor, being fully vortexed mixes, and then centrifuges 5min in 4 DEG C of 400 × g of use,
Retain the cell of bottom of the tube, add 300 μ l FACS buffer solutions and cell is resuspended, detected using flow cytometer.
Wild type SD rats testing result is as shown in Fig. 4-A；Lck knockout rat #1 testing results are as shown in Fig. 4-B；
Lck knockout rat #2 testing results are as shown in Fig. 4-C；As a result show, Lck gene knockout homozygote rats #1 and #2 are outside it
CD4 in all blood+T cell, CD8+T cell lacks completely, shows that we have successfully obtained Lck gene knockouts T cell missing
Rat model.
<110>Xinxiang College of Medical Science
<120>A kind of preparation method of SD rat T cells deleting genetic model
<160> 10
<170> PatentIn version 3.5
<211> 23
<212> DNA
<213>Sequence
<221>Lck genes sgRNA1
<400> 1
gcccaagtct ccatcatggg agg 23
<211> 23
<212> DNA
<213>Sequence
<221>Lck genes sgRNA2
<400> 2
gacccactgg tcacctatga ggg 23
<211> 64
<212> DNA
<213>Sequence
<221> Rat-LCK-IVT-1
<400> 3
ttaatacgac tcactatagg ggcccaagtc tccatcatgg ggttttagag ctagaaatag 60
caag 64
<211> 64
<212> DNA
<213>Sequence
<221> Rat-LCK-IVT-2
<400> 4
ttaatacgac tcactatagg ggacccactg gtcacctatg agttttagag ctagaaatag 60
caag 64
<211> 22
<212> DNA
<213>Sequence
<221> Rat-LCK-IVT-3
<400> 5
aaaaaagcac cgactcggtg cc 22
<211> 20
<212> DNA
<213>Sequence
<221> Lck-F
<400> 6
ccaggattcg gagctgttca 20
<211> 20
<212> DNA
<213>Sequence
<221> Lck-R
<400> 7
atttcctacc ccacagctgc 20
<211> 123
<212> DNA
<213>Sequence
<221>SgRNA1 double chain DNA sequences
<400> 8
ttaatacgac tcactatagg ggcccaagtc tccatcatgg ggttttagag ctagaaatag 60
caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt 120
ttt 123
<211> 123
<212> DNA
<213>Sequence
<221>SgRNA2 double chain DNA sequences
<400> 9
ttaatacgac tcactatagg ggacccactg gtcacctatg agttttagag ctagaaatag 60
caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt 120
ttt 123
<211> 358
<212> DNA
<213>Sequence
<221>Lck gene PCRs detect fragment
<400> 10
ccaggattcg gagctgttca cccttttcaa agcccaagtc tccatcatgg gagggctcat 60
agctgtgcag ggcaataact aggttgtctg taaagagaga aggagtcaag aggacccagc 120
ctggggctga caggaacagt gacgttctag agacacagga tgcagactaa gtggagagat 180
ggggaagagg gtatggacag gaacgggcaa gcctccacag tctgctgtta ttatgtttca 240
cacgctgtgg atggagtcac cttgcagcgg ggaggctggt gggagggagc cctcataggt 300
gaccagtggg tcccgaactt cagagccggt ccggatgggc agctgtgggg taggaaat 358
Claims (3)
- A kind of 1. preparation method of SD rat T cells deleting genetic model, it is characterised in that：Comprise the following steps：1) target sequence is determined：Two target sequences are respectively：sgRNA1：5 '-GCCCAAGTCTCCATCATGGG AGG-3 ', sgRNA2：5‘-GACCCACTGGTCACCTATGA GGG-3’；2) primer is designed：The sgRNA target sequences obtained according to step 1) design 3 primers, are respectively：Rat-LCK-IVT-1：5 '-TTAATACGAC TCACTATAGG GGCCCAAGTC TCCATCATGG GGTTTTAGAG CTAGAAATAG CAAG-3 ', Rat-LCK-IVT-2：5‘-TTAATACGAC TCACTATAGG GGACCCACTG GTCACCTATG AGTTTTAGAG CTAGAAATAG CAAG-3 ', Rat-LCK-IVT-3：5‘-AAAAAAGCAC CGACTCGGTG CC-3’；3) double chain DNA fragment is obtained：Rat-LCK-IVT-1 in step 2) and Rat-LCK-IVT-3 is matched, Rat-LCK-IVT- 1 and Rat-LCK-IVT-3 is matched, and using pX330 plasmids as template, enters performing PCR amplification；Pcr amplification product is purified, obtains double-strand DNA fragmentation；4) sgRNA mRNA are obtained：The double chain DNA fragment that step 3) is obtained is purified after transcribing in vitro as template, Obtain sgRNA1mRNA and sgRNA2mRNA；5) obtain Cas9mRNA and purified：Cas9 carriers are linearized through AgeI, purifies, produces after transcribing in vitro Cas9mRNA；6) SD rat T cells deleting genetic models are obtained：The Cas9mRNA that step 5) obtains is obtained with step 4) After sgRNA1mRNA and sgRNA2mRNA mixing, it is injected into rat fertilized egg cell, the fertilized oocyte of survival is entered False pregnancy SD rat female mice fallopian tubals；After false pregnancy SD rats are given a birth, Founder rats are obtained；Founder rats and wild type SD rats are returned, select F1Continue to be returned with wild type SD rats for heterozygote, repeat Heterozygote is picked out from offspring to be used to be returned, and heterozygote selfing is selected in backcross progeny, what self progeny obtained after testing Homozygote carries out Immunophenotype analysis, and complete T cell deficient is SD rat T cells deleting genetic models.
- 2. the preparation method of SD rat T cells deleting genetic model according to claim 1, it is characterised in that：Step 6) In, Cas9mRNA, sgRNA1mRNA, sgRNA2mRNA injection concentration are 50-100ng/ul.
- 3. the preparation method of SD rat T cells deleting genetic model according to claim 1, it is characterised in that：Step 6) It is middle progress Immunophenotype analysis concrete operations be：The homozygous peripheral blood of LCK gene knockout SD rats is extracted, passes through fluidic cell Art detects whether its T cell has lacked completely.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710646278.XA CN107446954A (en) | 2017-07-28 | 2017-07-28 | A kind of preparation method of SD rat T cells deleting genetic model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710646278.XA CN107446954A (en) | 2017-07-28 | 2017-07-28 | A kind of preparation method of SD rat T cells deleting genetic model |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107446954A true CN107446954A (en) | 2017-12-08 |
Family
ID=60490604
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710646278.XA Pending CN107446954A (en) | 2017-07-28 | 2017-07-28 | A kind of preparation method of SD rat T cells deleting genetic model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107446954A (en) |
Cited By (25)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108300738A (en) * | 2018-02-01 | 2018-07-20 | 新乡医学院 | A kind of preparation method of the humanized mouse model of the neutrophil leucocyte missing of NOD genetic backgrounds |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| CN112410341A (en) * | 2020-12-01 | 2021-02-26 | 新乡医学院 | Inducible neutrophil specific knockout mouse model construction method |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105624196A (en) * | 2015-12-24 | 2016-06-01 | 江苏大学 | Method for establishing CYP2C11 gene knockout rat model |
-
2017
- 2017-07-28 CN CN201710646278.XA patent/CN107446954A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105624196A (en) * | 2015-12-24 | 2016-06-01 | 江苏大学 | Method for establishing CYP2C11 gene knockout rat model |
Non-Patent Citations (3)
| Title |
|---|
| T. J. MOLINA 等: "Profound block in thymocyte development in mice lacking p56lck", 《NATURE》 * |
| 刘燕 等: "利用CRISPR／Cas9技术建立基因外显子敲除小鼠模型", 《中华老年医学杂志》 * |
| 田兰: "p56lck与T细胞发育", 《生命科学》 * |
Cited By (39)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
| US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| CN108300738A (en) * | 2018-02-01 | 2018-07-20 | 新乡医学院 | A kind of preparation method of the humanized mouse model of the neutrophil leucocyte missing of NOD genetic backgrounds |
| US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| CN112410341B (en) * | 2020-12-01 | 2023-05-23 | 新乡医学院 | Mouse model construction method capable of inducing specific elimination of neutrophils |
| CN112410341A (en) * | 2020-12-01 | 2021-02-26 | 新乡医学院 | Inducible neutrophil specific knockout mouse model construction method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107446954A (en) | A kind of preparation method of SD rat T cells deleting genetic model | |
| CN105039339B (en) | A kind of method of specific knockdown sheep FecB genes with RNA mediations and its special sgRNA | |
| CN103409468B (en) | A kind of establishment method of immunodeficient mouse model | |
| Farboud et al. | Enhanced genome editing with Cas9 ribonucleoprotein in diverse cells and organisms | |
| CN108660161A (en) | Method of the preparation based on CRISPR/Cas9 technologies without mosaic gene knock-out animal | |
| CN106318934A (en) | Carrot beta(1,2)xylosetransferase gene full sequence and plasmid construction of CRISPR (clustered regularly interspaced short palindromic repeats)/CAS9 for dicotyledon transfection | |
| CN104178461A (en) | CAS9-carrying recombinant adenovirus and application thereof | |
| CN109385451A (en) | One seed oyster CRISPR/Cas9 gene editing method | |
| CN106119284A (en) | A kind of product for building immunodeficient animals model and application thereof | |
| Gu et al. | Efficient generation of large‐fragment knock‐in mouse models using 2‐cell (2C)‐homologous recombination (HR)‐CRISPR | |
| CN106987604A (en) | A kind of method for preparing atherosclerosis disease model dog | |
| CN106755115A (en) | A kind of construction method of immunodeficient rats model | |
| CN108300738B (en) | Preparation method of humanized mouse model with NOD genetic background and neutrophilic granulocyte deletion | |
| CN109055434A (en) | A method of pig KIT gene structural mutation is corrected using CRISPRCas9 technology | |
| CN105950656A (en) | Method for rapidly obtaining gene knockout cell strains | |
| CN106282230A (en) | The method of rite-directed mutagenesis LDLR gene | |
| CN108251456A (en) | A kind of preparation method of the atherosclerosis mouse model of NOD genetic backgrounds | |
| CN105671045B (en) | A kind of method of sheep embryo fibroblast homologous recombination repair frequency after raising gene editing | |
| CN112410341A (en) | Inducible neutrophil specific knockout mouse model construction method | |
| CN106244556A (en) | The method of rite-directed mutagenesis ApoE gene | |
| CN109929876A (en) | The construction method of Vps28 knock out mice animal model and application | |
| CN109504708A (en) | A kind of gene targeting carrier and method of self deletion of selection markers | |
| CN109811009A (en) | A method of lethal gene function is analyzed using CRISPR/Cas9 system building knock-out mice model | |
| CN108949763A (en) | Accurate mutation LamR gene and the application of swine fever virus infection can effectively be inhibited | |
| CN103952442B (en) | The construction process of the nonimmune tolerance type OVA-HBsAg transgenic mice of Cre recombinase regulation and control |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171208 |
|
| RJ01 | Rejection of invention patent application after publication |